Bovine herpesvirus type 1 deletion mutants and vaccines

ABSTRACT

A deletion mutant of bovine herpesvirus type 1 which has a deletion in the glycoprotein gE-gene and which may further have a deletion in the thymidine kinase gene and/or the glycoprotein gI-gene, or have an insertion of a heterologous gene is disclosed. Recombinant nucleic acids which encode the gE-gene or a part thereof are also disclosed, in addition to vaccines and a method of treatment.

This application is a national phase 371 application of PCT/NL92/00097filed Jun. 5, 1992.

FIELD OF THE INVENTION

This invention relates to the fields of vaccination and diagnostics inconnection with diseases which are caused by pathogens and involves theuse of both the classic methods to arrive at a live attenuated vaccineor an inactivated vaccine and the modern methods based on DNArecombinant technology.

More specifically, the invention relates to live attenuated vaccines andinactivated vaccines for protecting animals, especially bovines, againstbovine herpesvirus type 1 (BHV-1), these vaccines being so designed thatthey are not only safe and effective, but also create the possibility ofdistinguishing infected from non-infected animals in a vaccinatedpopulation.

Diagnostic kits which can be used for such a test for distinguishinginfected from non-infected animals in a vaccinated population are alsoan aspect of the present invention.

BACKGROUND OF THE INVENTION

BHV-1, including the infections bovine rhinotracheitis virus (IBRV) andthe infectious pustular vulvovaginitis virus (IPVV), plays an importantrole in the development of respiratory diseases and fertility disordersin bovines. After an acute infection, BHV-1 often reminds present in thehost in a latent form. Latent virus can be reactivated under theinfluence of inter alia stress--which may or may not be accompanied byclinical phenomena--and subsequently excreted. As a consequence,infected cattle must be regarded as lifelong potential spreaders ofBHV-1. BHV-1 occurs endemically in an estimated 75% of Dutch cattlefarms. Especially older cattle are serologically positive.

There are a number of inactivated ("dead") vaccines and a number ofattenuated ("live") vaccines available for inoculation against BHV-1infections. Inactivated vaccines are prepared by killing the BHV-1virus, for instance by heat treatment, irradiation or treatment withethanol or formalin. However, these often give insufficient protection.Attenuated vaccines are prepared by a large number of passages onhomologous (bovine) or on heterologous cells such as porcine or caninecells, and sometimes viruses are also treated physically or chemicallythen. In this way, unknown mutations/deletions develop in the virusgenome, which often reduce the disease-producing properties of thevirus. Attenuated live vaccines give better protection than inactivatedvaccines, inter alia because they present more viral antigens to theimmune system of the host. Another important advantage of live vaccinesis that they can be administered intranasally, i.e., at the site wherethe first multiplication of the wild type virus occurs after infection.Yet, live vaccines leave room for improvement. Some live vaccines stillseem to possess their abortogenic ability, which becomes manifest inparticular after intramuscular administration. Moreover, probably alllive vaccines remain latently present in the vaccinated cow. Also, thereis a chance that if the vaccine differs only little from the wild-typevirus, reversion to virulence will occur. But one of the major problemsis that the BHV-1 vaccines cannot prevent infection by wild-typeviruses. The result is that vaccinated cattle can also spread wild-typeBHV-1.

For a proper BHV-1 control program, it is necessary to have disposal ofan efficacious and safe vaccine that can be distinguished from wild-typevirus, since the application of an efficacious vaccine can reduce thecirculation of BHV-1 considerably and a test which can distinguishbetween a vaccine and a wild-type virus makes it possible to detect (andthen remove) infected cattle in a vaccinated population.

Meanwhile, BHV-1 vaccines have been developed which seem to be saferthan conventional vaccines and are distinguishable from wild-type virus.A thymidine kinase deletion mutant has been isolated which isabortogenic to a lesser degree, becomes latent less frequently andcannot be reactivated. Further, using recombinant DNA techniques, aBHV-1 vaccine has been constructed which has a deletion in the gene forglycoprotein gIII, which makes this vaccine distinguishable fromwild-type BHV-1 by means of serological techniques. However, there arestill some objections to these vaccines. On the one hand, the thymidinekinase gene is involved in the viral replication and less replicationcan lead to less protection. On the other hand, the glycoprotein gIII isimportant for generating protective antibodies, which makes a gIIIdeletion vaccine less effective. A practical problem is that intranasaladministration, which generally gives the best protection, ofrecombinant vaccines is not allowed in some countries. Accordingly,there is a need for a vaccine which is safe as well as effective and yetcan be distinguished from wild-type BHV-1, it being further desirablethat at least one of such vaccines is based on a virus attenuated via aconventional route rather than a virus constructed by recombinant DNAtechniques.

Now, via passages in cell cultures, a BHV-1 strain has been obtainedwhich lacks the gene for glycoprotein gE. The first results of ourresearch indicate that this gene is quite useful to make a serologicaldistinction with regard to wild-type BHV-1 and that it is involved inthe expression of virulence. Therefore, its deletion contributes tosafety and may render the use of thymidine kinase deletions superfluous.The glycoprotein gE seems to be less important for induction ofprotection than the glycoprotein gIII. A conventionally attenuated BHV-1strain which can be serologically distinguished from wild-type virus isunique. The location and DNA sequence of the gE gene described hereinfor the first time were not previously known, nor were oligonucleotides,polypeptides and oligopeptides that can be derived therefrom. A test formaking a serological distinction on the basis of the gE gene is alsounique.

An important advantage of this "conventional" gE deletion mutant("conventional" refers to the use of a conventional method for isolatingan attenuated virus) is that it will be possible to administer itintranasally in countries where this is forbidden as far as recombinantvaccines are concerned. Taking due account of the different views onsafety, however, in addition to this conventional gE deletion vaccine,well-defined recombinant versions have been constructed as well. Theserecombinant vaccines also have a gE deletion--and may or may not have adeletion in the thymidine kinase gene as well--and can also be used asvectors for the expression of heterologous genes. All these recombinantvaccines can be distinguished from wild-type virus with the samegE-specific test. The use of a standard test for a set of differentvaccines can be a great advantage in the combat of BHV-1 as aninternational effort. Such an approach has not been previously describedin the field of BHV-1 vaccines.

Serological analysis of the anti BHV-1 response in cattle showed that animportant fraction of the anti-gE antibodies are directed against acomplex formed by glycoprotein gE and another BHV-1 glycoprotein:glycoprotein gI. Serological tests that can (also) demonstrate thepresence of such complex-specific antibodies may therefore be moresensitive than tests that can only detect anti-gE antibodies. Cattlevaccinated with a single gE deletion mutant may produce anti-gIantibodies that can interfere with the detection of anti-gI/gEantibodies. Consequently, this invention also includes a vaccine with agI/gE double deletion.

SUMMARY OF THE INVENTION

In the first place, this invention provides a deletion mutant of BHV-1which has a deletion in the glycoprotein gE-gene. The words "a deletionin" intend to cover a deletion of the gene as a whole.

A preferred embodiment of the invention is constituted by a deletionmutant of BHV-1 which has a deletion in the glycoprotein gE-gene whichhas been caused by an attenuation procedure, such as the deletion mutantDifivac-1 to be described hereinafter.

Other preferred embodiments of the invention consist of a deletionmutant of BHV-1 comprising a deletion in the glycoprotein gE-gene whichhas been constructed by recombinant DNA techniques, such as the deletionmutants 1B7 or 1B8 to be described hereinafter.

Another preferred embodiment of the invention consists of a doubledeletion mutant of BHV-1 comprising a deletion in the glycoproteingE-gene and a deletion in the glycoprotein gI-gene, such as the gI/gEdouble deletion mutant Difivac-IE to be described hereinafter.

Further, with a view to maximum safety, according to the invention adeletion mutant of BHV-1 is preferred which has a deletion in theglycoprotein gE-gene and a deletion in the thymidine kinase gene. Theinvention also covers a deletion mutant of BHV-1 which has a deletion inthe glycoprotein gE-gene, the glycoprotein gI-gene and the thymidinekinase gene.

The invention provides a vaccine composition for vaccination of animals,in particular mammals, more particularly bovines, to protect themagainst BHV-1, comprising a deletion mutant of BHV-1 as definedhereinabove, and a suitable carrier or adjuvant. Said composition may bea live or an inactivated vaccine composition.

The invention is further embodied in a mutant of BHV-1 which has adeletion in the glycoprotein gE-gene and contains a heterologous geneintroduced by recombinant DNA techniques. Preferably, this concerns amutant of BHV-1 which contains a heterologous gene introduced byrecombinant DNA techniques at the location of the glyoprotein gE-gene,which heterologous gene is under the control of regulatory sequences ofthe gE-gene and is optionally attached to the part of the gE-gene whichcodes for a signal peptide. Said heterologous gene may also be under thecontrol of a different promoter of BHV-1, or under the control of aheterologous promoter. When the mutant of BHV-1 has further deletions inaddition of the deletion in the glycoprotein gE-gene, such as a deletionin the thymidine kinase gene and/or a deletion in the glycoproteingI-gene, said heterologous gene may also be inserted at the location ofthis additional deletion(s). Plural insertions are another option,either together at the location of one deletion, or distributed overlocations of several deletions.

The heterologous gene introduced preferably codes for an immunogenicprotein or peptide of another pathogen, or for a cytokine which promotesthe immune response. Examples of suitable cytokines are interleukin 2,interferon-alpha and interferon-gamma.

The invention also provides a (live or inactivated) vaccine compositionfor vaccination of animals, in particular mammals, more particularlybovines, to protect them against a (different) pathogen, comprising amutant of BHV-1 having therein a heterologous gene coding for animmunogenic protein or peptide of that other pathogen, and a suitablecarrier of adjuvant. Of course, the protection may concern more than onepathogen, i.e. a multivalent vaccine wherein the mutant contains aplurality of heterologous genes.

The invention further relates to a composition comprising a recombinantnucleic acid comprising the glycoprotein gE-gene of BHV-1, a part ofthis glycoprotein gE-gene or a nucleotide sequence derived from thisglycoprotein gE-gene. This composition can contain a cloning orexpression vector having therein an insertion of a recombinant nucleicacid which comprises the glycoprotein gE-gene of BHV-1, a part of thisglycoprotein gE-gene or a nucleotide sequence derived from thisglycoprotein gE-gene.

The invention also comprises a composition comprising glycoprotein gE ofBHV-1, a part of this glycoprotein gE, a peptide derived from thisglycoprotein gE, or a complex of the glycoproteins gE and gI, and acomposition comprising an antibody which is specific for glycoprotein gEof BHV-1, a part of this glycoprotein gE, a peptide derived from thisglycoprotein gE, or a complex of the glycoproteins gE ad gI. "Antibody"is understood to mean both a polyclonal antibody preparation and amonoclonal antibody preferred for most applications. The terms "a partof glycoprotein gE" and "a peptide derived from glycoprotein gE" areunderstood to mean gE-specific amino acid sequences which generally willhave a length of at least about 8 amino acids.

The invention further relates to a diagnostic kit for detecting nucleicacid of BHV-1 in a sample, in particular a biological sample such asblood or blood serum, blood cells, milk, bodily fluids such as tears,lung lavage fluid, nasal fluid, sperm, in particular nervous tissue,coming from an animal, particularly a mammal, more particularly abovine, comprising a nucleic acid probe or primer having a nucleotidesequence derived from the glycoprotein gE-gene of BHV-1, and a detectionmeans suitable for a nucleic acid detection assay.

Further, the invention relates to a diagnostic kit for detectionantibodies which are specific for BHV-1, in a sample, in particular abiological sample such as blood or blood serum, saliva, sputum, bodilyfluid such as tears, lung lavage fluid, nasal fluid, milk, or tissue,coming from an animal, in particular a mammal, more in particular abovine, comprising glycoprotein gE of BHV-1, a part of this glycoproteingE, a peptide derived from this glycoprotein gE, or a complex of theglycoproteins gE and gI, and a detection means suitable for an antibodydetection assay. Such a diagnostic kit may further comprise one or moreantibodies which are specific for glycoprotein gE of BHV-1 or specificfor a complex of the glycoproteins gE and gI of BHV-1.

The invention also relates to a diagnostic kit for detecting protein ofBHV-1 in a sample, in particular a biological sample such as blood orblood serum, blood cells, milk, bodily fluids such as tears, lung lavagefluid, nasal fluid, sperm, in particular seminal fluid, saliva, sputumor tissue, in particular nervous tissue, coming from an animal, inparticular a mammal, more in particular a bovine, comprising one or moreantibodies which are specific for glycoprotein gE of BHV-1 or specificfor a complex of the glycoproteins gE and gI of BHV-1, and a detectionmeans suitable for a protein detection assay.

The invention further provides a method for determining BHV-1 infectionof an animal, in particular a mammal, more in particular a bovine,comprising examining a sample coming from the animal, in particular abiological sample such as blood or blood serum, blood cells, sperm, inparticular seminal fluid, saliva, sputum, bodily fluid such as tears,lung lavage fluid, nasal fluid, milk, or tissue, in particular nervoustissue, for the presence of nucleic acid comprising the glycoproteingE-gene of BHV-1, or the presence of the glycoprotein gE of BHV-1 or acomplex of the glycoproteins gE and gI of BHV-1, or the presence ofantibodies which are specific for the glycoprotein gE of BHV-1 orspecific for a complex of the glycoprotein gE and gI of BHV-1. Thesample to be examined can come from an animal which has not beenpreviously vaccinated with a vaccine composition according to theinvention or from an animal which has previously been vaccinated with avaccine preparation according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a set of BHV-1 vaccines, both live andinactivated, which have in common that they lack the glycoprotein gEgene in whole or in part. This set comprises both a natural gE deletionmutant and constructed gE deletion mutants which may or may not alsocomprise a deletion of the thymidine kinase gene and/or the glycoproteingI gene, and constructed gE deletion mutants which are used as vectorsfor heterologous genes. The invention further relates to nucleotidesequences encoding the BHV-1 glycoprotein gE-gene, oligonucleotidesderived from these sequences, the glycoprotein gE itself, peptides whichare derived therefrom and (monoclonal or polyclonal) antibodies whichare directed against the gE glycoprotein and peptides derived therefrom.The invention also relates to complexes of the glycoproteins gE and gIof BHV-1, and to antibodies directed against such complexes.

These materials according to the invention can be used for:

1) the vaccination of cattle against diseases caused by BHV-1, such thata distinction can be made between BHV-1 infected animals and vaccinatedanimals; the conventional and the constructed vaccine can be used sideby side;

2) the vaccination of cattle against both BHV-1 diseases and diseasescaused by other pathogens of which coding sequences for protectiveantigens can be incorporated into the BHV-1 deletion mutants;

3) testing blood, serum, milk or other bodily fluids from cattle todetermine serologically or by means of nucleic acid detection techniques(e.g. PCR) whether they have been infected by a wild-type BHV-1 or havebeen vaccinated with a gE deletion mutant. Synthesis of oligopeptides,polypeptides and glycoproteins derived from the coding sequence of theglycoprotein gE-gene and the glycoprotein gI-gene of BHV-1.

The results of the DNA sequence analysis, described in their examples,of the glycoprotein gE-gene (FIG. 3A-1 to 3A-6 SEQ ID NO.:1) and theisolated DNA fragments which code for this gene, make it possible, usingstandard molecular-biological procedures, both to synthesize peptides ofthe gE protein (oligo or polypeptides) and to express the gE protein inits entirety or in large parts via the prokaryotic route (in bacteria)or via the eukaryotic route (for instance in murine cells). Via theseroutes, gE-specific antigen can be obtained which can for instance servefor generating gE-specific monoclonal antibodies (Mabs). Furthermore,gE-specific antigen (and gE-specific Mabs) can be used in serologicaltests to enable a distinction to be made between animals vaccinated witha BHV-1 gE deletion vaccine and animals infected with wild-type BHV-1virus.

The results of the partial DNA sequence analysis of the glycoprotein gIgene--described in the examples--and the isolated DNA fragments thatcode for this gene, together with the eukaryotic cells expressingglycoprotein gE, allow the expression of the gI/gE complex in eukaryoticcells (See FIGS. 13 SEQ ID NO.:2 and 14 SEQ ID NO.:3 to SEQ ID NO.:6).This glycoprotein complex can be used to produce gI/gE specificmonoclonal antibodies. The gI/gE complex can also be used as antigen inserological tests to differentiate between cattle vaccinated with asingle gE BHV-1 deletion mutant or with a double gI/gE BHV-1 deletionmutant and cattle infected with wild type BHV-1 virus.

gE-specific peptides

On the basis of a known protein coding sequence, by means of anautomatic synthesizer, polypeptides of no less than about 40-50 aminoacids can be made. Now that the protein coding sequence of the gEglycoprotein of BHV-1 strain Lam has been unraveled (FIG. 3A SEQ IDNO.:1), polypeptides of this BHV-1 gE glycoprotein can be synthesized.With such polypeptides, according to standard methods, experimentalanimals such as mice or rabbits can be immunized to generate gE-specificantibodies. Further, using these gE-specific peptides, the locationswhere anti-gE antibodies react with the gE protein (the epitopes) can befurther specified, for instance with the PEPSCAN method (Geysen et al.,1984, Proc. Natl. Acad. Sci. USA 81, 3998-4002). gE-specificoligopeptides can also be used in serological tests which demonstrateanti-gE antibodies.

Prokaryotic expression of gE

For the synthesis of the gE protein in bacteria (i.e. the prokaryoticexpression of gE), DNA fragments which code for the glycoprotein gE orfor parts thereof must be cloned into prokaryotic expression vectors.Prokaryotic expression vectors are circular DNA molecules which canmaintain themselves in a bacterium as a separately replicating molecule(plasmid). These expression vectors contain one or more marker geneswhich code for an antibiotic resistance and thus enable the selectionfor bacteria with the expression vector. Further, expression vectorscomprise a (often controllable) promoter region behind which DNAfragments can be ligated which are then expressed under the influence ofthe promoter. In many current prokaryotic expression vectors, thedesired protein is expressed while fused to a so-called carrier protein.To that end, in the vector there is located behind the promoter thecoding sequence for the carrier protein, directly adjacent to which thedesired DNA fragment can be ligated. Fusion proteins are often morestable and easier to recognize and/or to isolate. The steady-state levelwhich a particular fusion protein can attain in a certain bacterialstrain differs from fusion to fusion and from strain to strain. It iscustomary to try different combinations. Eukaryotic expression of theglycoprotein gE-gene

Although prokaryotic expression of proteins offers some advantages, theproteins lack and modifications, such as glycosylation and the like,which occur in eukaryotic cells. As a result, eukaryotically expressedprotein is often a more suitable antigen. For the heterologousexpression of proteins in eukaryotic cells, such as murine cells, use ismade of eukaryotic expression vectors. These vectors are plasmids whichcan not only be multiplied in E. coli cells but also subsist stably ineukaryotic cells. In addition to a prokaryotic selection marker, therebyalso comprise a eukaryotic selection marker. Analogously to theprokaryotic expression vectors, eukaryotic expression vectors contain apromoter region behind which desired genes can be ligated. However, thepromoter sequences in eukaryotic vectors are specific for eukaryoticcells. Moreover, in eukaryotic vectors fusion to carrier proteins isutilized only rarely. These vectors are introduced into the eukaryoticcells by means of a standard transfection method (F. L. Graham and A. J.van der Eb, 1973, Virology 52, 456-467). In addition to the eukaryoticplasmid vectors, there are also viral vectors, where the heterologousgene is introduced into the genome of a virus (e.g. retroviruses,herpesviruses and vaccinia virus). Eukaryotic cells can then be infectedwith recombinant viruses.

In general, it cannot be predicted what vector and cell type are mostsuitable for a particular gene product. Mostly, several combinations aretried.

Eukaryotic expression of both the glycoprotein gE and the glycoproteingI

The final structure that a protein obtains, is depending on its primaryamino acid sequence, its folding, its posttranslational modificationsetc. An important factor that contributes to structure of a protein isits interaction with one or more other proteins. We have found that alsoBHV-1 glycoprotein gE forms a complex with at least one otherglycoprotein: BHV-1 glycoprotein gI. The first indication for such acomplex came from our results with candidate anti-gE Mabs 1, 51, 67, 75,and 78 (See table 2). These Mabs did not react with Difivac-1, nor withLam gE⁻ but also failed to recognize glycoprotein gE-expressing 3T3cells. However, these Mabs did react with gE-expressing 3T3 cells afterinfection with Difivac-1, showing that complementing factors are neededto give glycoprotein gE the proper antigenic conformation for theseMabs. In some of our radio-immunoprecipitation experiments with Mab 81we found coprecipitation of a protein with an apparent molecular weightof 63 kD. In view of the fact that the herpes simplex virus glycoproteingE forms a complex with a protein with a comparable molecular weight(HSV1 glycoprotein gI), we inferred that BHV-1 glycoprotein gE forms acomplex with the BHV-1 homolog of glycoprotein gI. To study this BHV-1gE/gI complex and to produce gE antigen with the proper antigenicstructure we expressed both glycoproteins in one eukaryotic cell. Forthis we applied the same procedures as described for the eukaryoticexpression of glycoprotein gE alone. The only additional prerequisite isthe use of expression vectors with different eukaryotic selectablemarkers.

Serological tests

Serological methods for making a distinction between cattle vaccinatedwith Difivac-1 and cattle infected with wild-type BHV-1 on the basis ofantibodies against gE are preferably based on the use of monoclonalantibodies directed against gE. These can be used in the followingmanners:

a) According to the principle described by Van Oirschot et al. (Journalof Virological Methods 22, 191-206, 1988). In this ELISA for thedetection of gI antibodies against the virus of Aujeszky's disease,antibodies are demonstrated by their blocking effect on the reaction oftwo Mabs having two different epitopes on gI. The test is carried out asfollows. Microtiter plates are coated with Mab 1, overnight at 37° C.,after which they are stored, e.g. at 4° C. or -20° C. The serum to beexamined is preincubated with antigen in separate uncoated microtiterplates, e.g. for 2 h at 37° C. The Mab 1-coated plates are washed, e.g.5 times, after which Mab 2 coupled to horseradish peroxidase (HRPO) isadded to these plates. Then the preincubated serum-antigen mixtures aretransferred to the plates in which the two Mabs are located, followed byincubation, e.g. for 1 h at 37° C. The plates are washed and substrateis added to each well. After e.g. 2 h at room temperature, the platesare spectrophotometrically read. Four negative control sera and fourserial dilutions of a positive serum are included on each plate. Theserum which has an optical density (OD) value of less than 50% of theaverage OD value of the 4 negative control sera which have been examinedon the same plate, is considered positive.

b) According to the Indirect Double Antibody Sandwich (IDAS) principle.Here, microtiter plates are coated with an Mab or a polyclonal serumdirected against the gE protein. Incubation with a gE-antigenpreparation results in gE binding to the coating. Antibodiesspecifically directed against gE in the bovine serum to be examinedsubsequently bind to the gE. These bound antibodies are recognized by ananti-bovine immunoglobulin conjugate. The antibodies in this conjugateare convalently bound to peroxidase enzyme. Finally, the bound conjugateis visualized by addition of a chromogenic substrate. The specificity ofthe reaction is checked by carrying out the same procedure with agE-negative control preparation instead of a gE-antigen preparation. Oneach microtiter plate, positive and negative control sera are included.The test is valid if the positive serum scores positive in a certaindilution. A serum is positive if it scores an OD which is 0.2 higherthan the standard negative control serum.

c) According to the IDAS principle as described under 2, but afterincubation of the serum to be examined an anti-gE Mab/HRPO is usedinstead of the anti-bovine immunoglobulin conjugate. An anti-gE peptideserum or an anti-gE polyclonal serum may be used instead of the anti-gEMab. The plates are washed and to each well a chromogenic substrate isadded. After e.g. 2 h room temperature, the plates arespectrophotometrically read. Four negative control sera and four serialdilutions of a positive serum are included on each plate. The serumwhich has an OD value of less than 50% of the average OD value of the 4negative control sera which have been examined on the same plate, isconsidered positive.

d) According to the principle of a blocking ELISA, whereby virus antigenwhich may or may not be purified is coated to the microtiter plateovernight. In these plates, the serum to be examined is incubated for,e.g. one hour or longer at 37° C. After a washing procedure, an anti-gEMab is added to the plates, followed by incubation for e.g. 1 h at 37°C. An anti-gE peptide serum or an anti-gE polyclonal serum may be usedinstead of the anti-gE Mab. The plates are washed and to each well achromogenic substrate is added. After e.g. 2 h at room temperature, theplates are read spectrophotometrically. Four negative control sera andfour serial dilutions of a positive serum are included on each plate.The serum which has an OD value of less than 50% of the average OD valueof the 4 negative control sera which have been examined on the sameplate, is considered positive.

In all the above arrangements, conventionally grown virus antigen whichcontains gE can be used, but so can gE-antigen which is expressed viaprokaryotes or eukaryotes. Alternatively, oligopeptides based on theBHV-1 gE sequence could be used in the above diagnostic tests instead ofconventional antigen. In addition, such oligopeptides could be used forthe development of a so-called "cow-side" test according to theprinciple described in an article of Kemp et al., Science 241,1352-1354, 1988. Such a test would then be based on a binding of theantigenic sequence of the oligopeptide by antibodies directed againstgE, present in infected animals. For such a test, the oligopeptide wouldhave to be coupled to an Mab directed against bovine erythrocytes.

Nucleic acid analysis using the polymerase chain reaction

Oligonucleotides (probes and primers) can for instance be used in thepolymerase chain reaction to make a distinction between vaccinated andinfected animals. The polymerase chain reaction (PCR) is a techniquewhereby nucleic acids of a pathogen can be multiplied billions of timesin a short time (De polymerase kettingreactie, P. F. Hilderink, J. A.Wagenaar, J. W. B. van der Giessen and B. A. M. an der Zeijst, 1990,Tijdscrift voor Diergeneeskunde deel 115, 1111-1117). The gEoligonucleotides can be chosen such that in a gE positive genome adifferent product is formed than in a gE negative genome. The advantageof this is that also an animal which has been vaccinated with a gEdeletion vaccine gives a positive signal in a PCR test. However, thisapproach depends on the presence of nucleic acids of the virus in asample, for instance blood, coming from the animal to be tested.

After an acute BHV-1 infection, there is a great chance that BHV-1specific nucleic acids can be demonstrated in the blood, but it has notbeen determined yet whether BHV-1 nucleic acids can also be demonstratedin the blood during latency.

The use of BHV-1 as a vector

For expressing heterologous genes in the BHV-1 genome, it is necessaryto have disposal of exact information of the area where the heterologousgene is to be inserted. There should not be any disturbance of essentialsequences, and regulatory sequences must be available for the expressionof the heterologous gene. In principle, the glycoprotein gE-gene is asuitable place to express heterologous genes. The gE-gene is notessential, hence there is no objection to replacing the gE gene by theheterologous gene. As a consequence, the heterologous gene can be sopositioned that it will be under the influence of the regulatorysequences of the gE gene. However, it is not necessary to use theregulatory sequences of the gE-gene. The expression of heterologousgenes may be controlled alternatively by other, e.g. stronger regulatorysequences of different genes. It is also possible to ligate theheterologous gene to the (export) signal peptide of the gE gene, so thatthe secretion of the heterologous gene product can be influenced. It isclear that detailed knowledge of the gE gene and the gE protein affordsthe possibility of using BHV-1 as a vector in a very measured manner.The vectors developed can moreover be serologically distinguished fromwild-type. The construction of BHV-1 mutants which express heterologousgenes can be carried out in the same manner as the constructed of gEdeletion mutants shown in the examples. However, the deletion fragmentsshould then be replaced with a fragment on which a heterologous gene islocated at the location of the deletion.

EXAMPLES

1) Isolation and identification of a natural gE deletion mutant

a) Isolation of a natural mutant

Genomic DNA was isolated from a number of conventionally attenuatedvaccines according to standard methods and analyzed using restrictionenzymes. In particular, we searched for genome deviations which would besuitable to enable distinction from wild-type BHV-1 virus.

Attention was directed in particular to the U_(S) region of the BHV-1genome, because in that region--by analogy with the herpes simplexvirus--probably a number of genes coding for non-essential glycoproteinare located Identification of a herpes simplex virus 1 glycoprotein genewithin a gene cluster dispensable for growth in cell culture, R.Longnecker, S. Chatterjee, R. J. Whitley and B. Roizman (1987) Proc.Natl. Acad. Sci. 84, 4303-4307!.

A batch of a BHV-1 vaccine coming from the University of Zagreb,Yougoslavia (Lugovic et al., Veterinarski Arhiv 55, 241-245, 1985),after a great number of passages on bovine embryonal kidney cells andembryonal bovine trachea cells (Ebtr), proved to have a deviant U_(S)region in addition to a normal U_(S) region. This vaccine moreoverappeared to form both large and small plaques on Ebtr cells. From thismixed population, a virus with a deviant U_(S) region was isolated bythree limiting dilution steps, with small plaques being chosen eachtime. The virus isolated via this route was examined further and calledDifivac-1, It has deposited with Institut Pasteur, Paris, France, on 27May 1992, deposit number I-1213.

Identification of the deletion of the gE gene in Difivac-1

For further analysis of this deviation in the U_(S) region, genomic DNAof Difivac-1 was isolated according to standard methods and subjected toSouthern blot analysis (FIG. 1A). Hybridization of this blot with a ³² Plabeled wild-type HindIII K fragment confirmed that this fragment,located centrally in the U_(S) region, is some 1.0 kilobase (kb) shorterin Difivac-1. Moreover, by this analysis, the position of the missingpart could be approximated (FIG. 1B). For further analysis of thedeletion, the U_(S) region of the wild-type BHV-1 strain Lam wasisolated and cloned into prokaryotic vectors. To that end, according tostandard methods, genomic DNA of the Lam strain (A of FIG. 2) wasisolated and cloned into the vectors pUC18, pACYC and pBR322 (B of FIG.2). A physical map of the area around the supposed position of thedeletion was composed (C of FIG. 2). Starting from this physical map,subclones suitable for the determination of the nucleotide sequence ofthis area were constructed in the vectors pKUN19 and pUC18 (D of FIG.2). Using these subclones, the nucleotide sequence of the two strands ofthe entire area (indicated in C of FIG. 2) was determined using theSanger method. This nucleotide sequence (SEQ ID NO.:1) was analyzedusing the PC/Gene program. From the conceptual translation, it appearedthat nucleotides (nt) 168 through nt 1893 code for an open reading frameof 575 amino acids (FIG. 3A-1 to 3A-6 SEQ ID NO.:1). Further analysisshowed that this amino acid sequence has the characteristics of atransmembrane glycoprotein as in shown in FIG. 3B. The fact is that thefirst 26 amino acids (aa) are recognized as a typically eukaryoticexport signal and the area between aa 423 and aa 450 is recognized as atransmembrane region. In addition, three potential N-bound glycosylationsites occur in this sequence. This predicted amino acid sequenceexhibits clear similarities to the glycoprotein gE-gene of herpessimplex virus (HSV); see FIGS. 4A and 4B SEQ ID NO.:7 to SEQ ID NO.:10.These and other similarities justify the conclusion that the gene foundis the gE homologue of BHV-1. For this reason, the gene is called gE. Todetermine to what extent this BHV-1 gE-gene is missing in Difivac-1, thep318 fragment was isolated. The p318 fragment starts on the AluI site 55nt before the postulated BHV-1 gE open reading frame and ends 133 ntbehind it. Genomic Difivac-1 DNA was analyzed with this p318 fragmentusing Southern blot hybridization. This revealed that Difivac-1 containsno p318 detectable sequences (FIG. 5). This experiment confirmed thatDifivac-1 contains a deletion and clearly demonstrates that thisdeletion extends throughout the entire gE gene.

To determine the size and the position of the deleted region, genomicsequences covering the U_(S) region of Difivac-1 were cloned intoprokaryotic vectors. See C of FIG. 11. The 14.5 kb EcoRI fragment wascloned into the pACYC vector and named p775. The 7.4 kb HindIII fragmentwas independently cloned into the pUC18 vector and named p728. Fromclone p728 two subclones were isolated: the 1.4 kb PstI fragment inclone p737 and the 350 bp AluI-PstI fragment in clone p754. Restrictionenzyme analysis and Southern blot analysis of these clones (data notshown), demonstrated that the gE deletion of Difivac-1 is 2.7 kb long,starting just 5' from the gE gene and ending at the border of the U_(S)region. These 2.7 kb have been replaced by a duplication of a 1 kbsegment, located in the U_(S) region opposite to the gE gene, as anaberrant extension of the repeat region. See B of FIG. 11. To confirmthe results of this analysis and to determine the exact recombinationpoint, the nucleotide sequence of most of the insert of clone p754 wasdetermined and compared with the wild type sequences. See FIG. 12 SEQ IDNO.:3 to SEQ ID NO.:6. This analysis showed that the recombination pointis located 77 bp upstream from the start codon of the gE gene.

c) Evaluation of safety and efficacy of Difivac-1

Difivac-1 was tested in BHV-1 seronegative specific pathogen free calvesof seven week old. Eight calves were intranasally vaccinated with 10⁵TCID₅₀ in 2 ml, of which 1 ml was sprayed in each nostril. Eight BHV-1seronegative specific pathogen free calves of seven week old, that werehoused in a separate isolation unit, were given 2 ml of culture mediumintranasally, and severed as unvaccinated controls. Five weeks aftervaccination, vaccinated and control calves were challenged intranasallywith 10⁷ TCID₅₀ of the highly virulent BHV-1 strain Iowa. Six weeksafter challenge all the calves were treated intramuscularly withdexamethasone for 5 days to reactivate putative latent virus. Clinicalsigns, rectal temperatures and body growth were monitored. Virusisolations were performed from nasal swabs, and neutralizing antibodytitres were determined in serum.

After vaccination, behaviour, appetite, rectal temperatures and growthrates of the calves remained normal, but the vaccinated calves had someserous nasal discharge and some hypersalivation. Lesions in nasal mucosawere not observed. Difivac-1 was excreted from nasal swabs aftervaccination (FIG. 17). All vaccinated calves produced neutralizingantibodies to BHV-1.

After challenge, all unvaccinated control calves showed apathy, loss ofappetite, ocular and nasal discharge, reddening of the gingiva of thelower jaw, severe lesions of the nasal mucosae until 14 days afterchallenge, and a growth arrest of 4 days. The vaccinated calves hadsmall, quickly healing lesions of the nasal mucosae and had no growtharrest. The daily clinical scores, the rectal temperature and growthdevelopment after challenge are given in FIGS. 18, 19 and 20. Afterchallenge, all calves shed virus from their nose, but the amount andperiod of virus excretion was markedly reduced in vaccinated calves(FIG. 21). A secondary antibody response developed in vaccinated calvesand the unvaccinated calves all produced antibodies after challenge.

After reactivation, the challenge virus was isolated from one vaccinatedcalf and from 5 unvaccinated calves. Difivac-1 could not be reactivated.

The above results demonstrate that Difivac-1 hardly induced any sign ofdisease in young calves and was not reactivated. Difivac-1 markedlyreduced the severity of disease and the amount of virus excretion afterchallenge.

In conclusion, Difivac-1 is a safe and efficacious vaccine for use incattle against BHV-1 infections.

2) Construction of recombinant gE deletion mutants of BHV-1

In order to be able to have disposal of differentiatable BHV-1 vaccineswhich are molecularly better defined than Difivac-1 and which, if sodesired, contain a deletion in for instance the thymidine kinase gene,in addition to a deletion in the gE gene, recombinant gE deletionmutants were constructed, in addition to Difivac-1. Starting from thedetermined position of the glycoprotein gE-gene and using the cloned DNAfragments which flank the gE-gene, a gE deletion fragment could beconstructed. Using a standard technique (F. L. Graham and A. J. van derEb, 1973, Virology 52, 456-467), this deletion fragment could berecombined in the genome of a wild-type BHV-1 strain, resulting in a gEdeletion mutant.

a) The construction of the gE deletion fragment

For the construction of the gE deletion fragment, a fragment was aimedfor which, on the other hand, lacks the entire gE sequence and, on theother, contains sufficient flanking sequence to allow recombination withthe wild-type genome. At the 5' (upstream) side, the 1.2 kb PstI-AsuIIfragment which ends 18 nt before the start codon of the gE gene waschosen. For the 3'(downstream) fragment the 1.2 kb EcoNI-DraI fragmentwas chosen, which starts 2 nt before the stop codon of the gE gene (FIG.6).

For the construction of the gE deletion fragment, the 1.4 kb PstI-SmaIfragment coming from the 8.4 kb HindIII K fragment of BHV-1 strain Lam,located at the 5' side of the gE gene, was subcloned into the SmaI andPstI site of plasmid pUC18.This clone was called p515. The EcoNI-SmaIfragment located on the 3' side of gE and coming from the 4.1 kbHindIII-EcoRI clone was cloned into their unique AsuII site of p515.Thus, the construction of the gE deletion fragment was completed and theclone so constructed was called p519. Although in principle the entirePstI-SmaI insert of p519 could be used as gE deletion fragment, this isnot advisable. The fact is that the PstI-SmaI extends approx. 100-150base pairs (bp) into the repeat sequence which flanks the U_(S) region.This piece of 100-150 bp could recombine with the repeat sequence on theother side of the U_(S) area where the gE gene is not located and couldthus yield undesirable recombination products. For that reason, thePstI-DraI fragment was chosen for the recombination experiment, so that100 bp of the repeat are removed.

b) Recombination of the gE deletion fragment with the genome ofwild-type BHV-1

In order to effect the recombination between the constructed gE deletionfragment and the genome of wild-type BHV-1, microgram amounts of the twoDNA molecules are cotransfected to Embryonal bovine trachea (Ebtr) cellsaccording to the standard method of F. L. Graham and A. J. van der Eb(1973, Virology 52, 456-467). Cellular recombination mechanisms lead tothe recombination of a small percentage of the DNA molecules (2-4%)which have been incorporated by the cells. For the selection of therecombined gE deletion mutants, the virus mixture that is formed aftertransfection is disseminated on a fresh Ebtr cell culture. In mostcases, the separate virus populations which thereby develop (plaques)originate from one virus. For the isolation of gE deletion mutants ofBHV-1 strain Lam, 230 of these plaques were isolated and examinedaccording to standard immunological methods with BHV-1 specificmonoclonal antibodies (Mabs) which do not react with Difivac-1 infectedcells. These Mabs are directed against the glycoprotein gE. Five of the230 plaques did not react with these Mabs. The DNA of these 5 plaqueswas further investigated.

c) DNA analysis of the constructed gE deletion mutants of BHV-1 strainLam

DNA preparations of 3 (1B7, 1B8 and 2H10) of the above mentioned 5candidate gE deletion mutants were further examined using the standardSouthern blot analysis technique (Sambrook et al. 1989). Doubledigestions of these DNA preparations with PstI and DraI, followed by gelelectrophoresis and Southern blot hybridization with the 2.3 kbPstI-DraI deletion fragment as probe show that the gE gene of the genomeof virus populations 1B7 and 1B 8 has been removed exactly in thedesired manner; see FIGS. 7A and 7B. Population 2H10 has a deviantPstI-DraI fragment. Southern blot hyridizations with a gE-specific probeshow that no gE sequences are located in any of the three DNApreparations (results are not shown). BHV-1 virus populations 1B7 and1B8 are intended recombinant gE deletion mutants. BHV-1 virus poplation1 B7 has been tested for vaccine properties.

d) Construction of thymidine kinase/gE double deletion mutants

Because BHV-1 recombinant deletion mutants with a deletion in only onegene may not be of sufficiently reduced virulence, deletions were alsoprovided in the thymidine kinase (TK) gene of the BHV-1 strains Lam andHarberink. These mutants were constructed in an analogous manner to thatuse for the above-mentioned gE deletion mutants (results are not shown).These TK deletion mutants have been used to construct TK/gE doubledeletion mutants.

e) Construction of glycoprotein gI/glycoprotein gE double deletionmutants

Because cattle vaccinated with a single gE deletion mutant may produceanti-gI antibodies that can interfere with the deletion of anti gI/gEantibodies (discussed below), we also invented a vaccine with a gI/gEdouble deletion. Such a gI/gE double deletion mutant can be constructedusing the same procedures used for the construction of the gE singledeletion mutant. Partial nucleotide sequence analysis of the upstreamend of the 1.8 kb PstI fragment--that covers the 5' end of the gEgene--revealed an open reading frame with significant homology to gIhomologs found in other herpesviruses. See FIGS. 13 SEQ ID NO.:2 and 14SEQ ID NO.:3 to SEQ ID NO.:6. Using the 350 bp SmaI-PstI fragment thatencompasses the putative 5' end of the gI gene and the EcoNI-SmaIfragment, located downstream of the gE gene, a gI/gE deletion fragmentcan be constructed. This fragment can be recombined with the wild typegenome to yield a BHV-1 gI/gE deletion mutant. See FIG. 16. The 80-90amino acids that--theoretically--may still be produced, will not be ableto elicit antibodies that can interfere with the detection of anti-gI/gEantibodies. Further sequence analysis of the gI gene will allow theconstruction of a gI deletion that covers the complete gI coding region.This gI/gE double deletion mutant has been named Difivac-IE.

f) Evaluation of safety and efficacy of the Lam gE⁻ and the Lam gE⁻, TK⁻mutants

Vaccine properties of the Lam gE⁻, and the Lam gE⁻, TK⁻ BHV-1 mutantstrains were tested in seven-week-old, BHV-1 seronegative, specificpathogen free calves. Each mutant strain was sprayed intranasally in 6calves. Each calf was given a total dose of 10⁵ TCID₅₀ in 2 ml culturemedium, of which 1 ml was sprayed in each nostril. Another 6 calves weresprayed intranasally with virus-free culture medium, and served asunvaccinated controls. Five weeks after vaccination all calves,vaccinated and controls, were challenged intranasally with 10⁷ TCID₅₀ ofthe highly virulent BHV-1 strain Iowa. After vaccination and afterchallenge, clinical signs, rectal temperatures and body weight weremonitored. Nasal swabs were taken to determine the number of days ofnasal virus shedding.

After vaccination, behaviour, appetite, rectal temperature and growthrates of the calves remained normal. Serous nasal discharge and smalllesions of the nasal mucosa were observed in all vaccinated calves.Virus could be isolated from the noses of the vaccinated calves forapproximately 7 days (Table 1).

After challenge, all unvaccinated control calves showed apathy, loss ofappetite, ocular and nasal discharge, reddening of the gingiva of thelower jaw, severe lesions of the nasal mucosae and growth was reduced.Calves vaccinated with Lam gE⁻, TK⁻ all developed some nasal dischargeand showed some minor lesions of the nasal mucosae. Not all calvesvaccinated with Lam gE⁻ did develop nasal discharge of lesions of thenasal mucosae. Apathy, loss of appetite, or other clinical symptoms ofdisease were not observed with vaccinated calves. Rectal temperature,growth and clinical score after challenge are shown in FIGS. 22, 23 and24. Unvaccinated calves shed virus from the nose 2 times longer thanvaccinated calves (Table 1).

The above results demonstrate that the Lam gE⁻ and the Lam gE⁻, TK⁻BHV-1 mutant strains hardly induced any clinical sign of disease inyoung calves. Both mutant strains prevented sickness after challenge andreduced the period of nasal virus shedding with 50%.

Lam gE⁻ and Lam gE⁻, TK⁻ BHV-1 mutant strains are safe and efficaciousfor use as a vaccine in cattle against BHV-1 infections.

3) Prokaryotic expression of gE

For the prokaryotic expression of the BHV-1 glycoprotein gE-gene, so faruse has been made of pGEX expression vectors (D. B. Smith and K. S.Johnson, Gene 67 (1988) 31-40). pGEX vectors code for the carrierprotein glutathione S-transferase (GST) from Schistosoma japonicum whichis under the influence of the tac promoter which can be induced toexpression by Isopropylthioglactoside (IPTG). An example of a GST-gEfusion protein is the product of construct pGEX-2T600s3 (FIG. 8A). Inthis construct, using standard molecular-biological techniques (Sambrooket al. 1989), a 600 bp SamI fragment which codes for an N-terminalregion of 200 amino acids of the gE protein was ligated behind the GSTgene. This construct was designed in triplicate, with each time adifferent reading frame of the 600 bp fragment being ligated to the GST.All three constructs were introduced into Escherichia coli strain DH5α,induced with IPTG and the proteins formed were transferred tonitrocellulose after polyacrylamide gel electrophoresis by means ofWestern blotting. Immunological detection with anti-GST antibodiesdemonstrated that only the proper reading frame (No. 3) which codes forthe gE protein area leads to the expression of a prominent fusionprotein of the predicted size of 27 k (GST)+20 k (gE)=47 k. Three of theMabs isolated y us that do not react with Difivac-1 recognize the 47 kDGST-gE fusion protein in a Western blot; see FIG. 8B.

4) Eukaryotic expression of the glycoprotein gE-gene

For the eukaryotic expression of the glycoprotein gE-gene, hetertoforeinter alia the vector pEVHis has been chosen. The pEVHis vector has, aseukaryotic marker, the HisD gene coding for the histidinol dehydrogenaseEC 1.1.1.23! (C. Hartmann and R. Mulligan, 1988, Proc. Natl. Acad. Sci.USA 85, 8047-8051) which causes cells to survive the toxic concentrationof 2.5 mM histidinol. The vector moreover comprises the promoter regionof the immediate early gene of the human cytomegalovirus (HCMV), withunique restriction enzyme sites located behind it. For the constructionof a pEVHis/gE expression vector, use was made of a fragment comprisingthe entire coding region of the glycoprotein gE-gene. It starts on theAluI site 55 bp before the postulated open reading frame of gE and ends133 bp behind it. This region was cloned behind the HCMV promoter of thepEVHis vector, whereby the construct pEVHis/gE was formed (FIG. 9). ThepEVHis/gE was amplified in E. coli DH5α cells and purified by means of acesium chloride gradient (Sambrook et al., 1989). This purified DNA wastransfected to Balb/C-3T3 cells according to the method of Graham andVan der Eb. Transformed cells were selected with histidinol, whereaftertwenty histidinol resistant colonies could be isolated. These colonieswere examined with Mab 81 by means of an Immuno Peroxidase MonolayerAssay (IPMA). Four colonies proved to express the gE protein. Of thesefour colonies, 3T3 gE clone 9 was used to isolate a subclone having ahigh gE expression. The clone isolated by this method (called 3T3gE 9.5)was used for characterizing candidate anti-gE monoclonal antibodies.

5) Eukaryotic expression of both the BHV-1 glycoprotein gE and the BHV-1glycoprotein gI in the same cell

To express the BHV-1 glycoprotein gI in the same cell as the BHV-1glycoprotein gE we first determined the putative position of the BHV-1gIgene. Because the herpes simplex virus glycoprotein gI gene is locatedjust upstream of the glycoprotein gE gene, it was inferred that theBHV-1 gI gene would be located on a corresponding position. To testthis, the sequence has been determined of a region of 283 nucleotides,located about 1 kb upstream of the start of the BHV-1 gE gene.Conceptual translation of this region showed that the second readingframe codes for a 94 amino acids sequence that is homologous to theherpes simplex virus glycoprotein gI (FIGS. 13 SEQ ID NO.:2 and 14 SEQID NO.:6). Because the homologous segment is about 80 amino acids fromthe start codon the putative start of the open reading frame of theBHV-1 gI gene is estimated about 250 nt upstream from the sequencedregion. From this it was inferred that the 1.7 kb SmaI fragment thatstarts 400 nt upstream from the sequenced region and ends within the gEgene should contain the complete coding region of the BHV-1 gI gene. The1.7 kb SmaI fragment has been cloned into the eukaryotic vector MSV-neo(See FIG. 15). This vector contains the strong Murine Sarcoma Viruspromoter and the selector gene neo that codes for the resistance againstthe antibiotic G-418 sulphate Geneticin. The resulting constructMSVneoGI has been amplified in E. coli DH5α cells and has beentransfected into 3T3gE 9.5 cells using the method of Graham and Van derEb. The transfected cells were selected with 400 μg Geneticin/ml culturemedium and the resistant colonies have been isolated and tested withcandidate anti-gE Mabs that failed to react with 3T3gE 9.5 cells. Fromthis we selected the 3T3gE/gI R20 clone that reacted with e.g. Mab 66 aswell as wild type BHV-1 does.

6) Characterization of candidate anti-gE Mabs

Mabs were produced against wild-type BHV-1 and selected for theirinability to react with Difivac-1 infected embryonic bovine trachea(Ebtr) cells. These Mabs are examined for their reactivity with

a) the Lam gE⁻ deletion mutant;

b) the above described prokaryotic expression product in a Western blot;

c) the above described gE-expressing Balb/c-3T3 cells;

d) cells mentioned under c) and infected with Difivac-1, and

e) Balb/c-3T3 cells expressing the gE/gI complex.

For reactivity testing under a, c, d and e an immunoperoxidase monolayerassay (IPMA) was used. The results in Table 2 show that we have producedMabs that are directed against gE (nrs. 2, 3, 4, 52, 66, 68, 72 and 81)and Mabs (nrs. 1, 51, 53, 67, 75 and 78) that may be directed againstconformational antigenic domains on the gE/gI complex. A competitionIPMA to map the antigenic domains recognized by the various Mabsindicated that at least 4 antigenic domains are present on glycoproteingE and that one domain probably is formed by the gE/gI complex (Table2).

Detection of anti-gE antibodies in cattle infected by BHV-1

To examine whether in serum of infected cattle antibodies against gE arepresent an indirect blocking IPMA was performed with the 16 candidategE-Mabs and the following 8 selected sera:

2 sera of bovines vaccinated with Difivac-1 and challenged with thevirulent Iowa strain, that were collected 14 days after challenge;

2 sera of bovines experimentally infected with BHV-1 subtype 1 virus,that were collected 20 months after infection. One of the bovines wasinfected by contact exposure;

2 sera of bovines experimentally infected with BHV-1 subtype 2b virus,that were collected 20 months after infection. One of the bovines wasinfected by contact exposure;

a serum of a specific pathogen free calf vaccinated with a ts mutantvaccine and challenged 3 weeks later with BHV-1 subtype 2b virus, thatwas collected 7 weeks after challenge;

a serum of a gnotobiotic calf vaccinated with a ts mutant vaccine andchallenged 3 weeks later with BHV-1 subtype 2b virus, that was collected7 weeks after challenge.

Table 2 shows that all these sera contained antibodies against theantigenic domains III and IV on gE, and against antigenic domain I thatis probably located on the gE/gI complex. We may conclude that gEappears to be a suitable serological marker to distinguish betweenBHV-1-infected and vaccinated cattle.

7) Detection of BHV-1 nucleic acids by means of the PCR procedure usingBHV-1 gE-specific primers

Starting from the determined nucleotide sequence of the BHV-1 gE gene, aprimer pair suitable for the PCR was selected, using the primerselection program by Lowe et al. (T. Lowe, J. Sharefin, S. Qi Yang andC. W. Dieffenbach, 1990, Nucleic Acids Res. 18, 1757-1761). Theseprimers were called P₃ and P₄ and are shown in FIG. 10 SEQ ID NO.:1. Theprimers are located 159 nt apart and lead to the amplification of afragment of 200 nt. Using primers P₃ SEQ ID NO.:15 and P₄ SEQ ID NO.:16and isolated BHV-1 DNA, the conditions for the PCR procedure wereoptimized. This involved in particular the variation of the MgCl₂concentration, the glycerol concentration and the cycling conditions.The optimum buffer found for the use of P₃ SEQ ID NO.:15 and P₄ SEQ IDNO.:16 for the amplification of BHV-1 DNA is 10 mM Tris pH 8.0, 50 mMKCl, 0.01% gelatin, 2.6 mM MgCl₂ and 20% glycerol. The optimum cyclicconditions found (Perkin Elber Cetus DNA Thermal Cycler) are for cycli1-5: 1 min. 98° C., 30 sec. 55° C. and 45 sec. 72° C. and for cycli6-35: 30 sec. 96° C., 55° C. and 45 sec. 72° C. After the PCRamplification, the 200 nt DNA fragment obtained was electrophoresed on a2% agarose gel, blotted on nitrocellulose and subsequently subjected toSouthern blot analysis. The ³² P dCTP labeled probe used for theSouthern blot analysis is the 137 bp TaqI fragment which is locatedbetween the primer binding sites (FIG. 10 SEQ ID NO.:14). Afterautoradiography of the hybridized filters, a 200 bp band can beobserved. Via this route, amplification of only 10 BHV-1 genomes(approx. 1.5×10⁻¹⁵ μg DNA) still leads to a property detectable signal(result not shown). In a comparable manner, a PCR procedure wasdeveloped using primers which are based on the coding sequence of theBHV-1 glycoprotein gIII (D. R. Fitzpatrick, L. A. Babiuk and T. Zamb,1989, Virology 173, 46-57). To enable a distinction to be made betweenwild-type BHV-1 DNA and a gE deletion mutant vaccine, DNA samples weresubjected both to the gE-specific PCR and to gIII-specific PCR analysis.In such a test, a Difivac-1 DNA preparation was found to be gIIIpositive and gE negative. Because the detection of BHV-1 DNA in bovinesemen will be an important use of the BHV-1 specific PCR procedure, itwas attempted to perform the gE-specific PCR on bovine semen infectedwith BHV-1. However, unknown components in the semen have a stronglyinhibitory effect on the polymerase chain reaction. Therefore, aprotocol was developed to isolate the BHV-1 DNA from bovine semen. Toisolate the DNA from bovine semen, 30 μl of semen is incubated with 1mg/ml proteinase K (pK) in a total volume of 300 μl 0.15M NaCl, 0.5%Na-Sarkosyl and 40 mM DTT, at 60° C. After 1 hour the sample is allowedto cool down to room temperature and 300 μl 6M NaI is added andincubated for 5 min. From this mixture DNA is isolated with a standardchloroform/isoamylethanol extraction and precipitated with 1 volumeisopropanol. The precipitate is washed with 2.5M NH₄ Ac/70% ethanol andresuspended in 10 mM Tris pH 7.4, 1 mM EDTA, 0.5% Tween 80 and 0.1 mg/mlpK for a second incubation for 1 hour at 60° C. This DNA preparation canbe directly submitted to the Polymerase Chain Reaction.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B:

Southern blot analysis of BHV-1 strains Difivac-1 and Iowa

FIG. 1A. Drawing of an autoradiogram of a Southern blot of Difivac-1 andIowa genomic DNA. In lanes 1 and 3, Difivac-1 DNA was applied afterrestriction enzyme digestion with HindIII and PstI, respectively. Inlanes 2 and 4, Iowa DNA was applied after restriction enzyme digestionwith HindII and PstI, respectively. The size of the fragments isindicated in kilobase (kb).

Viral DNA was isolated by centrifuging the culture medium (70 ml/rollerbottle of ca. 450 cm²) with virus infected Ebtr cells for 2 h through a25% (w/w) sucrose cushion, in 10 mM Tris pH 7.4, 150 mM NaCl and 1 mMEDTA at 20 krpm in the SW27 rotor of the Beckman L5-65 ultracentrifuge.From the virus pellet so obtained, DNA was isolated according tostandard methods (J. Sambrook, E. F. Fritsch and T. Maniatis, 1989,Molecular cloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, New York). On this DNA, restriction enzyme digestionswere performed with enzymes from Boehringer Mannheim in the SuRE/cutbuffers supplied by the manufacturer.

After separation on a 0.7% agarose gel for horizontal electrophoresisand blotting on a nitrocellulose filter (Schleicer & Schuell, Inc.) thefilter was prehybridized for 6 h at 42° C. in 50% foramide, 3×SSC(1×SSC=0.15M NaCl and 0.015M Na-citrate, pH 7.4), 50 μl denatured salmonsperm DNA (Sigma)/ml and 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone and 0.02% ficoll and 0.1% Na-dodecylsulphate (SDS). Then,hybridization was performed by adding to the same solution the ³² P dCTP(Amersham) labeled HindIII K fragment (The choice of the HindIII Kfragment is based on: Cloning and cleavage site mapping of DNA frombovine herpesvirus 1 (Copper strain), John F. Mayfield, Peter J. Good,Holly J. VanOort, Alphonso R. Campbell and David A. Reed, Journal ofVirology (1983) 259-264). After 12-14 h hybridization, the filter waswashed for 2 h in 0.1% SDS and 0.1×SSC at 60° C. The HindIII K fragmentwas cloned into the pUC18 vector according to standard cloningprocedures (J. Sambrook, E. F. Fritsch and T. Maniatis, 1989, molecularcloning: a laboratory manual, 2nd ed. Cold Spring Harbor LaboratoryPress, New York). After HindIII digestion of the pUC/8.4 HindIIIK clonethe pUC18 vector was separated from the 8.4 kb HindIII K fragment againby electrophoresis on a 0.7% Low Melting Point Agarose (BRL, LifeTechnologies, Inc.) gel, and isolated from the agarose by standardphenol extraction and ethanol precipitation. The isolated HindIII Kfragment was labeled with the Random Primed DNA labeling Kit 1004.760from Boehringer Mannheim. Autoradiography of the hybridized filters wascarried out through 36 h exposition of a Kodak XAR film at -70° C.,using a reflecting screen.

FIG. 1B. Physical maps of the 8.4 kb HindIII K fragment of Iowa and ofthe 7.4 kb HindIII fragment of Difivac-1. In view of the comigration ofthe 6 kb PstI fragments and the absence of the 1.8 kb PstI fragment inDifivac-1, the deletion is postulated in the hatched area.

FIG. 2:

Subcloning of wild-type BHV-1 fragments around the region lacking inDifivac-1

In A the components of the BHV-1 genome are shown: The Unique Long(U_(L)) region; the Unique Short (U_(S)) region and the two repeats (Irand Tr). This map is based on the published analysis of the Cooperstrain (John F. Mayfield, Peter J. Good, Holly J. VanOort, Alphonso R.Campbell and David A. Reed, Journal of Virology (1983) 259-264).

In B the fragments are shown from the U_(S) region which have beencloned into prokaryotic vectors: A 15.2 kb EcoRI fragment in pACYC, an8.4 kb HindIII fragment in pUC18 and a 2.7 kb and a 4.1 kb EcoRI-HindIIIfragment in pBR322. The isolation of the viral DNA fragments was carriedout according to the procedures which are mentioned in the legends ofFIG. 1A. The cloning of these fragments into the various vectors wascarried out according to standard procedures (J. Sambrook, E. F. Fritschand T. Maniatis, 1989, Molecular cloning: a laboratory manual, 2nd ed.Cold Spring Harbor Laboratory Press, New York).

In C a physical map is shown of the region where the postulated deletionin Difivac-1 is localized.

In D some subclones of this region are indicated, which were used forfurther analysis. The two PstI fragments were cloned into pKUN19 and theremaining fragments into pUC18.

FIGS. 3A and 3B:

FIGS. 3A-1 to 3A-6 SEQ ID NO.:1, Nucleotide sequence of 2027 nucleotidesfrom the U_(S) region of BHV-1 strain Lam around the postulated locationwhich has been deleted in Difivac-1, as indicated in C of FIG. 2 fromthe AluI recognition site on the extreme left to the HincII recognitionsite on the extreme right!. The nucleotide sequence in the inserts ofthe subclones shown in D of FIG. 2 was determined by analyzing on thetwo strands using the dideoxy sequence method of Sanger et al. (F.Sanger, S. Nicklen and A. R. Coulson, 1977, Proc. Natl. Acad. Sci. USA74, 5463-5467). To that end, the T7 sequence kit of Pharmacia was usedaccording to the procedure specified by the manufacturer. For theradioactive labeling, ³⁵ S! dATP (Amersham) was used. The sequenceanalysis of the GC rich regions with compression artefacts was repeatedwith the 7-deaza-dGTP variant of the Pharmacia kit. Indicated beneaththe nucleotide sequence is, in the three-letter code, the amino acid(aa) sequence of the open reading frame of 575 aa residues, which wasfound after conceptual translation of the nucleotide sequence. Thistranslation is based on the universal code and was determined using thePC/gene computer program (PC/gene version 1.03, November 1987). Thisopen reading frame of 575 aa starts with the methionine at nt 168 andends with the stop codon at nucleotide 1893.

Structural analysis of the open reading frame of 575 aa residues wasalso performed with the PC/gene computer program. The first 26 aa from aeukaryotic export signal indicated in the figure by "signal peptide".With a score of 6.2, the cleavage of this signal sequence is predictedbetween aa 26 and aa 27. The sequence of 575 aa as 3 possible N-boundglycosylation sites (NXT/S) indicated by a line under the amino acidresidues. According to the Rao and Argos method there is a transmembraneregion between aa 423 and aa 450 indicated in the figure by"transmembrane helix". Recognition sequences (sites) for the restrictionenzymes AsuII, SmaI, HindIII and EcoNI are underlined. The calculatedmolecular weight of this polypeptide is 61212.

FIG. 3B. Schematic representation of the structural characteristics ofthe above mentioned 575 aa open reading frame.

FIGS. 4A and 4B:

Amino acid comparison of the amino acid sequence of the BHV-1 gE genewith the amino acid sequence of the herpes simplex virus (HSV) gE geneand other gE homologous genes pseudo-rabies virus (PRV) gI andvaricella-zoster (VZV) gpI!

The sequences used for this comparison come from the followingpublications; HSV: Sequence determination and genetic content of thesort unique region in the genome of herpes simplex virus type 1. D. J.McGeoch, A. Dolan, S. Donald and F. J. Rixon (1985) Journal Mol. Biol.181, 1-13. VZV: DNA sequence of the U_(S) component of thevaricella-zoster virus genome. A. J. Davidson (1983), EMBO Journal 2,2203-2209. PRV: Use of λgt11 to isolate genes for two pseudorabies virusglycoproteins with homology to herpes simplex virus and varicella-zostervirus glycoproteins. E. A. Petrovskis, J. G. Timmins and L. E. Post(1986) Journal of Virology 60, 185-193!. These sequences were comparedusing the sequence analysis program Multalin (F. Corpet, 1988, Nucl.Acids Res. 16, 10881-10890).

In FIG. 4A a diagram shown in which all four amino acid sequences areshown schematically. Here, the predicted transmembrane parts (TM) areshown below each other. In addition to the predicted export signalsequences (SP) and the possible N-bound glycosylation sites (I), twoconserved areas are shown, in which the relative position of thecysteine residues is often unchanged (C C C).

In FIG. 4B SEQ ID NO.:7 to SEQ ID NO.:14 the results are shown of theMultalin comparison on the centrally located cysteine rich region of thefour gE versions. Asterisks indicate identical amino acids and colonsanalogous amino acids.

FIG. 5:

Drawing of photographs obtained in a Southern blot analysis of Difivac-1and Iowa

Panel A: Genomic DNA of Difivac-1 and Iowa restriction enzyme digestionswith BstI (1,2), EcoRI (3,4) and HindIII (5,6) separated on a 0.7%agarose gel, blotted on nitrocellulose and hybridized with ³² P labeledHindIII K fragment of BHV-1 strain Lam according to the procedurespecified in the legends of FIG. 1A.

Panel B: Nitrocellulose blot of the same gel as in A hybridized with theBHV-1 gE-specific probe p318. This probe comprises the entireAluI-HincII region indicated in FIG. 2C.

FIG. 6:

Construction of gE deletion fragment BHV-1

In A the position of the gE gene and the clones used is shown. Thecomponents of the BHV-1 genome are: The Unique Long (U_(L)) region; theUnique Short (U_(S)) region and the two repeats (IR and TR). To obtainthe region located on the 5' side of the gE gene, the 1.4 kb PstI-SmaIfragment from the 8.4 kb HindIII K fragment of BHV-1 strain Lam wassubcloned into the SmaI and PstI site of plasmid pUC18. This clone wascalled p515 and is shown in B. The EcoNI-SmaI fragment located on the 3'side of gE, coming from the 4.1 kb HindIII-EcoRI clone was cloned intothe unique AsuII site of p515. To enable the ligation of the EcoNI restto the AsuII rest, clone p515 was digested with AsuII, then treated withKlenow enzyme (Boehringer Mannheim) and dCTP to provide one cytosineresidue in the AsuII rest according to standard methods (Sambrook etal., 1989). This additional cytosine is indicated by an asterisk i D.Then, p515 was also digested with the SmaI enzyme, whereafter the EcoNIfragment could be ligated into this vector. The clone thus constructedwas called p519.

FIGS. 7A and 7B:

FIG. 7A. Drawing of a photograph obtained in Southern blot analysis ofDNA preparations of 1B7, 1B8 and 2H10. DNA isolation, restriction enzymedigestions, blotting and hybridization were performed according to theprocedures described in the legends of FIG. 1A. After PstI-DraI doubledigestion of the DNA preparations 1B7, 1B8 and 2H10, the fragments wereseparated on a 0.7% agarose gel and subsequently blotted on anitrocellulose filter. This filter was hybridized with the ³² P dCTPlabeled 2.3 kb PstI-DraI deletion fragment as probe. In lanes 1 through3, the samples 1B7, 1B8 and 2H10 were separated, respectively. In lane4, wild-type BHV-1 DNA of the Lam strain was applied and in lane 5 the2.3 kb deletion fragment.

FIG. 7B Physical map of the 15.2 kb EcoRI fragment of BHV-1 strain Lam.The map shows the position of the PstI, DraI and HindIII recognitionsites and the position of the hybridization probe mentioned in thedescription of FIG. 7A.

FIGS. 8A and 8B:

Prokaryotic expression of BHV-1 gE

For the prokaryotic expression of BHV-1 gE, the 600 bp SmaI fragment ofthe gE gene was fused in three reading frames to the coding region ofthe glutathione-S-transferase gene from Schistosoma japonicum in thevector pGEX-2T (D. B. Smith and K. S. Johnson, Gene 67 (1988) 31-40).Recombinant molecules with the proper (syn) orientation of the SmaIfragment were identified by means of restriction enzyme analysis usingstandard methods. E. coli DH5α clones with this fusion construct werecalled pGEX-2T600s1, pGEX-2T600s2 and pGEX-2T600s3.

FIG. 8A: Diagram of one of the pGEX-2T600s constructs. Located on theNH₂ side of the region which codes for GST-gE fusion product is theIsopropylthiogalactoside (IPTG) inducible tac promoter region.

FIG. 8B. Drawing of photographs obtained in Western blot analysis oftotal protein preparations of DH5α cells transformed with pGEX-2T600s.Overnight cultures of DH5α cells transfected with the constructspGEX-2T600s1, pGEX-2T600s2 and pGEX-2T600s3 were continued 1/10 inLuria-Bertani (LB) medium with 50 μg/ml ampicillin and after 1 h growthinduced with IPTG for 5 h. These induced cultures were centrifuged for 5min at 6,000×g and incorporated in 1×layermix (2% SDS, 10% Glycerol, 5%mecaptoethanol and 0.01% bromophenol blue) 1.5 ml culture isincorporated in 500 μl layermix! and heated at 95° C. for 5 min. Then 50μl per lane was separated on a vertical 12.5% polyarcrylamide gelaccording to standard procedures and subsequently Semi-dry blotted to anitrocellulose filter using the LKB-multiphor II Nova Blot system underthe conditions specified by the manufacturer.

In lanes M, prestained marker protein was applied (BRL LifeTechnologies, Inc. 236 k, 112 k, 71 k, 44 k, 28 k, 18 k and 15 k) and inlanes 1, 2 and 3 the total protein preparations of DH5α cellstransfected with the three respective frames: pGEX-2T600s1, pGEX-2T600s2and pGEX-2T600s3.

In panel A, the result can be seen of the western blot analysis withanti-GST serum. To that end, the filter was incubated according tostandard procedures (E. Harlow and D. Lane, 1986, Antibodies: alaboratory manual, Cold Spring Harbor Laboratory, New York) in blockingbuffer (PBS+2% milk powder and 0.05% Tween 20) and subsequently withpolyclonal anti-GST rabbit serum. Then the filter was washed andincubated with horse radish peroxidase (HRPO) conjugatedgoat-anti-rabbit immunoglobulin serum. Then the bound goat antibodieswere immunochemically detected with chromogen (diaminobenzidine,chloronaphthol and H₂ O₂). The GST fusion product which is indicated byan arrow has the predicted size of approx. 47 k only in frame 3.

In panel B, the result can be seen of the western blot analysis withmonoclonal antibody Mab 4, which recognizes the gE protein. To that end,a duplo filter as in panel A was blocked, incubated with Mab, washed,and incubated with HRPO conjugated rabbit-anti-mouse serum. Then, thebound rabbit antibodies were immunochemically detected with chromogen.The band which is visible in lane 3 (frame 3) is 47 k in size and isindicated by an arrow.

FIG. 9:

Construction of the pEVHisgE plasmid for the eukaryotic expression ofthe BHV-1 gE gene

For the eukaryotic expression of the gE gene, the entire gE codingregion was cloned in the proper orientation behind the HCMV promoterregion of the expression vector pEVHis using standard procedures(Sambrook et al. 1989). To that end, the 394 bp AluI fragment whichstarts 55 bp before the open reading frame of the gE was cloned intopUC18 and called p201. Then, after HincII digestion of p201, the 1740 bpHincII fragment, which comprises the greater part of the gE gene, wascloned into p201. This resulted in the plasmid p318 which in thepolylinker of pUC18 comprises the entire gE coding area from the AluIsite 55 bp before the start codon of gE to the HincII site 133 bp behindthe stop codon of gE. Using the restriction enzyme sites in thepolylinker of the vector, this fragment was cut from p 318 with theenzymes BamHI and SphI. First, 318 was digested with SphI and then theSphI site was filled in using Klenow polyerase and dNTP's. After thedigestion with BamHI, the 1.9 kb insert was separated from the pUC18vector in Low Melting Point Agarose and ligated in the pEVHis vectorwhich had been digested with BamHI and EcoRV to that end. The plasmid toformed was called pEVHis/gE.

FIG. 10:

Position of the gE-specific primers and probe for the PCR procedure fordetecting BHV-1 DNA

Shown in the Figure is the nucleic acid sequence SEQ ID NO.:14 of theBHV-1 glycoprotein gE gene from nucleotide 1272 to 2027 the sequence hasbeen taken over from FIG. 3A-1 to 3A-6!. The primers used for thegE-specific PCR procedure were called P₃ and P₄. The primer bindingsites for P₃ and P₄ are underlined. The nucleotide sequence of P₃ is5'-ACG-TGG-TGG-TGC-CAG-TTA-GC-3' (SEQ ID NO.:15). The nucleotidesequence of P₄ is (complementary to the primer binding sequencespecified above) 5'-ACC-AAA-CTT-TGA-ACC-CAG-AGC-G-3' (SEQ ID NO.:16).The probe which was used for the Southern blot hybridization for thedetection of the PCR amplified DNA, is the 137 bp TagI fragment locatedbetween the primer binding sites See SEQ ID NO.:14, the ends of thisfragment being indicated. For comparison with FIG. 3A-1 to 3A-6 SEQ IDNO.:1, the HindIII and the EcoNI sites are also indicated.

FIG. 11:

Mapping of the gE deletion of Difivac-1

A shows the physical map of the 15.5 kb EcoRI fragment of the wild typeBHV-1 strain Lam. B shows the physical map of the 14.5 kb EcoRI fragmentof Difivac-1. Both EcoRI fragments cover the complete Unique shortregions of the genomes of the respective viruses. The position of the gEgene and the putative position of the gI gene have been indicated byopen boxes. Maps A and B are positioned in such a way, that the 6 kbPstI fragments within each map are aligned. In both maps the internalrepeat and the terminal repeat sequences have been indicated by hatchedboxes. The arrows beneath the repeats indicate the orientation of thesesequences.

In A the part of the U_(S) region that is missing in the Difivac-1strain has been indicated.

C shows the position of the cloned Difivac-1 fragments used to map thegE deletion and to obtain the physical map shown in B. The arrowsbeneath the inserts of clones p728, p737 and p754 indicate the regionsthat have been sequenced to determine the recombination point.

Abbreviations

A=AluI, E=EcoRI, P=PstI, H=HindIII, r=recombination point, IR=internalrepeat, TR=terminal repeat.

FIG. 12:

Determination of the exact recombination point in the U_(S) region ofDifivac-1

To determine the exact borders of the gE deletion found in the Difivac-1strain, clone p754 and the ends of clones p728 and p737 have beensequenced. The inserts of these clones have been indicated in FIG. 11.The sequence procedures used have been described in the legends of FIGS.3A-1 to 3A-6.

In A SEQ ID NO.:11 the sequence of most of the AluI--PstI fragment hasbeen shown. This sequence starts in the promoter region of the gE gene.A putative TATA box has been underlined. At point r (=recombinationpoint) this promoter region is fused to a sequence also found at theopposite site of the U_(S) region, named: inverted repeat. The exactrecombination point has been determined by comparing the repeat found atthe gE promoter region with the copy of the repeat found at the oppositesite of the U_(S) region. The point were these sequences diverge hasbeen indicated in B(I) SEQ ID NO.:12 with `r`. A similar comparison hasbeen made with the gE promoter sequence fund in Difivac-1 and the gEpromoter found in wild type strain Lam. The point were these sequencesdiverge has been shown in B(II) SEQ ID NO.:13 and also indicated with`r`. The recombination points found are the same.

FIG. 13:

Partial sequence analysis of the BHV-1 gI gene

Using the 1.8 kb PstI clone of BHV1 strain Lam, that reaches into boththe BHV-1 gI and gE gene (See FIG. 11), the sequence of 284 nucleotideswithin the coding region of BHV-1 gI was determined SEQ ID NO.:2. Thesequence procedure used have been described in the legends of FIG. 3A.The sequence has been translated based on the universal code by thePC/gene computer program version 1.03 (Nov. 1987). The amino acidsequence encoded by the second reading frame is given in the one lettercode beneath the nucleotide sequence. This amino acid sequence ishomologous to the coding region of other herpes virus gI homologs (SeeFIG. 14 SEQ ID NO.:3 to SEQ ID NO.:6).

FIG. 14:

Amino acid comparison of the partial amino acid sequence of the putativeBHV-1 gI gene SEQ ID NO.:3 with the corresponding parts of the codingregions of the herpes simplex virus (HSV1) gI gene SEQ ID NO.:5, thepseudorabies virus (PRV) gp63 gene SEQ ID NO.:4, and thevaricella-zoster virus (VZV) gpIV gene SEQ ID NO.:6.

The PRV sequence SEQ ID NO.:4 starts at amino acid 82, the HSV1 sequencestarts at aa 80 SEQ ID NO.:5 and the VZV sequence starts at aa 76 SEQ IDNO.:6 of their respective coding regions. The sequences used werepublished in the papers mentioned in the legends of FIG. 4. Thecomparison was performed using the Multalin computer program. Asterisksindicate identical amino acids and colons indicate analogous aminoacids.

FIG. 15:

Construction of the MSVneoGI plasmid for the eukaryotic expression ofthe BHV-1 gI gene

Based on the amino acid comparisons of the partial sequence of the BHV-1gI gene SEQ ID NO.:3 the putative position of the BHV-1 gI gene SEQ IDNO.:3 has been estimated. Based on this estimation it was inferred thatthe 1.7 kb SmaI fragment should contain the complete coding region ofthe BHV-1 gE gene. The position of this 1.7 kb SmaI fragment has beenindicated in A. To the blunt ends of this 1.7 kb SmaI fragment, BamHIlinkers have been ligated, using standard procedures. The resultingproduct was digested with BamHI and ligated into the eukaryoticexpression vector MSV-neo. The MSV-neo vector has a unique BamHI sitebehind the MSV-LTR, which has a strong promoter activity. This vectorhas been described in Rijsewijk et al., 1987 EMBO J. 6, 127-131.

FIG. 16:

Construction of a BHV-1 gI/gE double deletion fragment

The position of the glycoprotein gE gene and the putative position ofthe glycoprotein gI gene in the U_(S) region of BHV-1 are depicted indiagram A. The hatched blocks indicate the repeats that border the U_(S)region. B shows the physical map of some essential restriction enzymesites with respect to the position of both genes. To construct the gI/gEdeletion fragment clone p1.7-SmaI/o containing the 1.7 kb SmaI fragmentthat embraces the gI gene will be digested with PstI. The PstI site ofthe remaining 350 bp SmaI-PstI insert will be made blunt ended usingstandard molecular biological procedures. The EcoNI-SmaI fragment (see Bof FIG. 6), isolated from the 4.1 kb HindIII-EcoRI fragment described inA of FIG. 6, will also be made blunt ended and ligated to the modifiedPstI site. This is diagrammed in C and D. From the resulting clone pΔIEthe 1.4 kb SmaI-DraI fragment can be isolated to recombine with wildtype BHV-1 DNA.

Abbreviations

E=EcoRI, H=HindIII, S=SmaI, P=PstI, ENI=EcoNI, D=DraI, kb=kilobase andU_(S) =unique short.

FIG. 17:

Mean nasal virus shedding from calves after vaccination ·=vaccinatedwith Difivac-1, 0=Unvaccinated control.

FIG. 18:

Mean daily clinical score of calves after challenge with a virulentBHV-1 strain, key as in FIG. 17.

FIG. 19:

Mean rectal temperature of calves challenge with a virulent BHV-1strain, key as in FIG. 17.

FIG. 20:

Mean growth of calves after challenge with a virulent BHV-1 strain, keyas in FIG. 17.

FIG. 21:

Mean nasal virus shedding from calves after challenge with a virulentBHV-1 strain, key as in FIG. 17.

FIG. 22:

Mean rectal temperature of calves after challenge with a virulent BHV-1strain ·=vaccinated with Lam gE⁻, 0=vaccinated with Lam gE⁻ /TK⁻,x=unvaccinated control.

FIG. 23:

Mean growth of calves after challenge with a virulent BHV-1 strain, keyas in FIG. 22.

FIG. 24:

Mean daily clinical score of calves after challenge with a virulentBHV-1 strain, key as in FIG. 22.

                  TABLE 1                                                         ______________________________________                                        Nasal virus shedding of calves after vaccination with Lam gE.sup.-            or Lam gE.sup.- /TK.sup.- and after challenge with a virulent BHV-1           strain                                                                        of these vaccinated and control calves                                                  Average number of days of nasal virus shedding                      Group         After vaccination                                                                         After challenge                                     ______________________________________                                        Control       0           10.33 ± 1.51                                     Lam gE.sup.-  7.00 ± 0.89                                                                            4.83 ± 1.17                                      Lam gE.sup.- /TK.sup.-                                                                      7.17 ± 1.33                                                                            5.17 ± 0.98                                      ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Characterization of gE-Mabs                                                   REACTIVITY OF CANDIDATE gE-Mabs WITH                                               Difi-                                                                         vac-1                     3T3 gE                                              3T3/    Lam               Difi- 3T3  Ag    Ab                            Mab  EBTR    gE.sup.-                                                                             Prok.                                                                              3T3 gE                                                                              vac-1 gE/gI                                                                              group cattle                        ______________________________________                                        1    -       -      nd   -     +     ?    I     +                             2    -       -      -    +     +     +    II    -                             3    -       -      +    +     +     +    ?     -                             4    -       -      +    +     +     +    ?     -                             42   -       -      nd   -     -     ?    V?    ±                          51   -       -      nd   -     +     +    III   +                             52   -       -      +    +     +     +    ?     -                             53   -       -      nd   -     +     +    III   +                             59   -       -      nd   -     -     +    III   +                             66   -       -      nd   +     +     +    III   +                             67   -       -      nd   -     +     +    III   +                             68   -       -      -    +     +     +    IV    +                             72   -       -      -    +     +     +    V     ±                          75   -       -      nd   -     +     ?    I     +                             78   -       -      nd   -     +     ?    nd    -                             81   -       -      -    +     +     +    II?   -                             ______________________________________                                         +: All 8 tested sera score a blocking percentage of >50% in an indirect       blocking IPMA.                                                                ±: Sera score a blocking percentage of ±50%.                            -: Sera score a blocking percentage of <50%.                             

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2027 nucleotides                                                  (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AGGGCGGAGCGTTGAGCGGCCCGACCGCCGCCGGGTTGTTAAATGGGTCT50                          CGCGCGGCTCGTGGTTCCACACCGCCGGAGAACCAGCGCGAGCTTCGCTG100                         CGTGTGTCCCGCGAGCTGCGTTCCGGGGAACGGCGCACGCGAGAGGGTTC150                         GAAAAGGGCATTTGGCA167                                                          ATGCAACCCACCGCGCCGCCCCGGCGGCGGTTGCTGCCGCTGCTGCTG215                           MetGlnProThrAlaProProArgArgArgLeuLeuProLeuLeuLeu                              151015                                                                        CCGCAGTTATTGCTTTTCGGGCTGATGGCCGAGGCCAAGCCCGCGACC263                           ProGlnLeuLeuLeuPheGlyLeuMetAlaGluAlaLysProAlaThr                              202530                                                                        GAAACCCCGGGCTCGGCTTCGGTCGACACGGTCTTCACGGCGCGCGCT311                           GluThrProGlySerAlaSerValAspThrValPheThrAlaArgAla                              354045                                                                        GGCGCGCCCGTCTTTCTCCCAGGGCCCGCGGCGCGCCCGGACGTGCGC359                           GlyAlaProValPheLeuProGlyProAlaAlaArgProAspValArg                              505560                                                                        GCCGTTCGCGGCTGGAGCGTCCTCGCGGGCGCCTGCTCGCCGCCCGTG407                           AlaValArgGlyTrpSerValLeuAlaGlyAlaCysSerProProVal                              65707580                                                                      CCGGAGCCCGTCTGCCTCGACGACCGCGAGTGCTTCACCGACGTGGCC455                           ProGluProValCysLeuAspAspArgGluCysPheThrAspValAla                              859095                                                                        CTGGACGCGGCCTGCCTGCGAACCGCCCGCGTGGCCCCGCTGGCCATC503                           LeuAspAlaAlaCysLeuArgThrAlaArgValAlaProLeuAlaIle                              100105110                                                                     GCGGAGCTCGCCGAGCGGCCCGACTCAACGGGCGACAAAGAGTTTGTT551                           AlaGluLeuAlaGluArgProAspSerThrGlyAspLysGluPheVal                              115120125                                                                     CTCGCCGACCCGCACGTCTCGGCGCAGCTGGGTCGCAACGCGACCGGG599                           LeuAlaAspProHisValSerAlaGlnLeuGlyArgAsnAlaThrGly                              130135140                                                                     GTGCTGATCGCGGCCGCAGCCGAGGAGGACGGCGGCGTGTACTTCCTG647                           ValLeuIleAlaAlaAlaAlaGluGluAspGlyGlyValTyrPheLeu                              145150155160                                                                  TACGACCGGCTCATCGGCGACGCCGGCGACGAGGAGACGCAGTTGGCG695                           TyrAspArgLeuIleGlyAspAlaGlyAspGluGluThrGlnLeuAla                              165170175                                                                     CTGACGCTGCAGGTCGCGACGGCCGGCGCGCAGGGCGCCGCGCGGGAC743                           LeuThrLeuGlnValAlaThrAlaGlyAlaGlnGlyAlaAlaArgAsp                              180185190                                                                     GAGGAGAGGGAACCAGCGACCGGGCCCACCCCCGGCCCGCCGCCCCAC791                           GluGluArgGluProAlaThrGlyProThrProGlyProProProHis                              195200205                                                                     CGCACGACGACACGCGCGCCCCCGCGGCGGCACGGCGCGCGCTTCCGC839                           ArgThrThrThrArgAlaProProArgArgHisGlyAlaArgPheArg                              210215220                                                                     GTGCTGCCGTACCACTCCCACGTATACACCCCGGGCGATTCCTTTCTG887                           ValLeuProTyrHisSerHisValTyrThrProGlyAspSerPheLeu                              225230235240                                                                  CTATCGGTGCGTCTGCAGTCTGAGTTTTTCGACGAGGCTCCCTTCTCG935                           LeuSerValArgLeuGlnSerGluPhePheAspGluAlaProPheSer                              245250255                                                                     GCCAGCATCGACTGGTACTTCCTGCGGACGGCCGGCGACTGCGCGCTC983                           AlaSerIleAspTrpTyrPheLeuArgThrAlaGlyAspCysAlaLeu                              260265270                                                                     ATCCGCATATACGAGACGTGCATCTTCCACCCCGAGGCACCGGCCTGC1031                          IleArgIleTyrGluThrCysIlePheHisProGluAlaProAlaCys                              275280285                                                                     CTGCACCCCGCCGACGCGCAGTGCAGCTTCGCGTCGCCGTACCGCTCC1079                          LeuHisProAlaAspAlaGlnCysSerPheAlaSerProTyrArgSer                              290295300                                                                     GAGACCGTGTACAGCCGGCTGTACGAGCAGTGCCGCCCGGACCCTGCC1127                          GluThrValTyrSerArgLeuTyrGluGlnCysArgProAspProAla                              305310315320                                                                  GGTCGCTGGCCGCACGAGTGCGAGGGCGCCGCGTACGCGGCGCCCGTT1175                          GlyArgTrpProHisGluCysGluGlyAlaAlaTyrAlaAlaProVal                              325330335                                                                     GCGCACCTGCGTCCCGCCAATAACAGCGTAGACCTGGTCTTTGACGAC1223                          AlaHisLeuArgProAlaAsnAsnSerValAspLeuValPheAspAsp                              340345350                                                                     GCGCCGGCTGCGGCCTCCGGGCTTTACGTCTTTGTGCTGCAGTACAAC1271                          AlaProAlaAlaAlaSerGlyLeuTyrValPheValLeuGlnTyrAsn                              355360365                                                                     GGCCACGTGGAAGCTTGGGACTACAGCCTAGTCGTTACTTCGGACCGT1319                          GlyHisValGluAlaTrpAspTyrSerLeuValValThrSerAspArg                              370375380                                                                     TTGGTGCGCGCGGTCACCGACCACACGCGCCCCGAGGCCGCAGCCGCC1367                          LeuValArgAlaValThrAspHisThrArgProGluAlaAlaAlaAla                              385390395400                                                                  GACGCTCCCGAGCCAGGCCCACCGCTCACCAGCGAGCCGGCGGGCGCG1415                          AspAlaProGluProGlyProProLeuThrSerGluProAlaGlyAla                              405410415                                                                     CCCACCGGGCCCGCGCCCTGGCTTGTGGTGCTGGTGGGCGCGCTTGGA1463                          ProThrGlyProAlaProTrpLeuValValLeuValGlyAlaLeuGly                              420425430                                                                     CTCGCGGGACTGGTGGGCATCGCAGCCCTCGCCGTTCGGGTGTGCGCG1511                          LeuAlaGlyLeuValGlyIleAlaAlaLeuAlaValArgValCysAla                              435440445                                                                     CGCCGCGCAAGCCAGAAGCGCACCTACGACATCCTCAACCCCTTCGGG1559                          ArgArgAlaSerGlnLysArgThrTyrAspIleLeuAsnProPheGly                              450451460                                                                     CCCGTATACACCAGCTTGCCGACCAACGAGCCGCTCGACGTGGTGGTG1607                          ProValTyrThrSerLeuProThrAsnGluProLeuAspValValVal                              465470475480                                                                  CCAGTTAGCGACGACGAATTTTCCCTCGACGAAGACTCTTTTGCGGAT1655                          ProValSerAspAspGluPheSerLeuAspGluAspSerPheAlaAsp                              485490495                                                                     GACGACAGCGACGATGACGGGCCCGCTAGCAACCCCCCTGCGGATGCC1703                          AspAspSerAspAspAspGlyProAlaSerAsnProProAlaAspAla                              500505510                                                                     TACGACCTCGCCGGCGCCCCAGAGCCAACTAGCGGGTTTGCGCGAGCC1751                          TyrAspLeuAlaGlyAlaProGluProThrSerGlyPheAlaArgAla                              515520525                                                                     CCCGCCAACGGCACGCGCTCGAGTCGCTCTGGGTTCAAAGTTTGGTTT1799                          ProAlaAsnGlyThrArgSerSerArgSerGlyPheLysValTrpPhe                              530535540                                                                     AGGGACCCGCTTGAAGACGATGCCGCGCCAGCGCGGACCCCGGCCGCA1847                          ArgAspProLeuGluAspAspAlaAlaProAlaArgThrProAlaAla                              545550555560                                                                  CCAGATTACACCGTGGTAGCAGCGCGACTCAAGTCCATCCTCCGCTAG1895                          ProAspTyrThrValValAlaAlaArgLeuLysSerIleLeuArg*                                565570575                                                                     GCGCCCCCCCCCCCCCGCGCGCTGTGCCGTCTGACGGAAAGCACCCGCGT1945                        GTAGGGCTGCATATAAATGGAGCGCTCACACAAAGCCTCGTGCGGCTGCT1995                        TCGAAGGCATGGAGAGTCCACGCAGCGTCGTC2027                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 284 nucleotides                                                   (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CTACCACGCCGCGGGCGACTGCTTCGTTATGCTGCAGACGACCGCGTT48                            TyrHisAlaAlaGlyAlaCysPheValMetLeuGlnThrThrAlaPhe                              51015                                                                         CGCCTCCTGCCCGCGCGTCGCGAACGACGCCTTTCGCTCCTGCCTGCA96                            AlaSerCysProArgValAlaAsnAspAlaPheArgSerCysLeuHis                              202530                                                                        CGCCGACACGCGCCCCGCTCGCAGCGAGCGGCGCGCGAGCGCCGCGGT144                           AlaAspThrArgProAlaArgSerGluArgArgAlaSerAlaAlaVal                              354045                                                                        CGAAAACCACGTGCTCTTCTCCATCGCCCATCCGCGCCCAATAGACTC192                           GluAsnHisValLeuPheSerIleAlaHisProArgProIleAspSer                              505560                                                                        AGGGCTCTACTTTCTGCGCGTCGGCATCTACGGCGGCACCGCGGGCAG240                           GlyLeuTyrPheLeuArgValGlyIleTyrGlyGlyThrAlaGlySer                              65707580                                                                      CGAGCGCCGCCGAGACGTCTTTCCCTTGGCCGCGTTTGTACACA284                               GluArgArgArgAspValPheProLeuAlaAlaPheValHis                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrHisAlaAlaGlyAspXaaCysPheValMetLeuGlnThrThrAla                              51015                                                                         PheAlaSerCysProArgValAlaAsnXaaAlaPheArgSerCysLeu                              202530                                                                        HisAlaAspThrArgProXaaAlaArgSerGluArgArgAlaSerAla                              354045                                                                        AlaValGluAsnHisValLeuPheSerIleAlaHisProArgProIle                              505560                                                                        AspSerGlyLeuTyrPheLeuArgValGlyIleTyrGlyGlyXaaThr                              65707580                                                                      AlaGlySerGluArgArgArgAspValPheProLeuAlaAlaPheVal                              859095                                                                        His                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 98 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ArgLeuAspProLysArgAlaXaaCysTyrThrArgGluTyrAlaAla                              51015                                                                         GluTyrAspLeuCysProArgValHisHisGluAlaPheArgGlyCys                              202530                                                                        LeuArgXaaXaaXaaLysArgXaaGluProLeuAlaArgArgAlaSer                              354045                                                                        AlaAlaValGluAlaArgArgLeuLeuPheValSerArgProAlaPro                              505560                                                                        ProAspAlaGlySerTyrValLeuArgValArgXaaXaaAsnGlyXaa                              65707580                                                                      ThrThrAspLeuPheValLeuThrAlaLeuValProProArgGlyArg                              859095                                                                        ProHis                                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TyrProMetGlyHisLysXaaCysProArgValValHisValValThr                              51015                                                                         ValThrAlaCysProArgArgProAlaValAlaPheAlaLeuCysArg                              202530                                                                        AlaThrAspSerThrHisXaaSerProAlaTyrProThrLeuGluLeu                              354045                                                                        AsnLeuAlaGlnGlnProLeuLeuArgValGlnArgAlaThrArgAsp                              505560                                                                        TyrAlaGlyValTyrValLeuArgValTrpValGlyAspAlaProAsn                              65707580                                                                      AlaSerLeuPheValLeuGlyMetAlaIleAlaAlaGluGly                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrAlaAspThrValAlaPheCysPheArgSerValGlnValIleArg                              51015                                                                         TyrAspGlyCysProArgIleArgThrSerAlaPheIleSerCysArg                              202530                                                                        TyrLysHisSerTrpHisTyrGlyAsnSerThrAspArgIleSerThr                              354045                                                                        GluProAspAlaGlyValMetLeuLysIleThrLysProGlyIleAsn                              505560                                                                        AspAlaGlyValTyrValLeuLeuValArgLeuAspHisSerArgSer                              65707580                                                                      ThrAspGlyPheIleLeuGlyValAsnValTyrThrAlaGly                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       HisSerGlnLeuPheSerProGlyAspThrPheAspLeuMetProArg                              51015                                                                         ValValSerAspMetGlyAspSerArgGluAsnThrPheThrAlaThr                              202530                                                                        LeuAspTrpTyrTyrAlaArgAlaProProArgCysLeuLeuTyrTyr                              354045                                                                        ValTyrGluProCysIleTyrHisProArgAlaProGluCysLeuArg                              505560                                                                        ProValAspProAlaCysSerPheThrSerProAlaArgAlaAlaLeu                              65707580                                                                      ValAlaArgArgAlaTyrAlaSerCysSerProLeuLeuGlyAspArg                              859095                                                                        TrpLeuThrAlaCysProPheAspAlaPheGlyGluGluValHisXaa                              100105110                                                                     XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaThrAsnAlaThr                              115120125                                                                     AlaAspGluSerGlyLeuTyrValLeuValMetThrHisAsnGlyHis                              130135140                                                                     ValAlaThrTrpAspTyrThrLeuValAlaThr                                             145150155                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       HisSerHisValPheSerValGlyAspThrPheSerLeuAlaMetHis                              51015                                                                         LeuGlnTyrLysIleXaaHisXaaXaaGluAlaProPheAspLeuLeu                              202530                                                                        LeuGluTrpLeuTyrValProIleAspProThrCysGlnProMetArg                              354045                                                                        LeuTyrSerThrCysLeuTyrHisProAsnAlaProGlnCysLeuSer                              505560                                                                        HisMetAsnSerGlyCysThrPheThrSerProHisLeuAlaGlnArg                              65707580                                                                      ValAlaSerThrValTyrGlnAsnCysXaaXaaGluHisAlaAspAsn                              859095                                                                        TyrThrAlaTyrCysLeuGlyIleSerHisMetGluProSerPheGly                              100105110                                                                     LeuIleLeuHisAspGlyGlyThrThrLeuLysPheValAspThrPro                              115120125                                                                     GluSerLeuSerGlyLeuTyrValPheTyrValTyrPheAsnGlyHis                              130135140                                                                     ValGluAlaValAlaTyrThrValValSerThr                                             145150155                                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       HisSerHisValTyrThrProGlyAspSerPheLeuLeuSerValArg                              51015                                                                         LeuGlnSerGluPhePheAspXaaXaaGluAlaProPheSerAlaSer                              202530                                                                        IleAspTrpTyrPheLeuArgThrAlaGlyAspCysAlaLeuIleArg                              354045                                                                        IleTyrGluThrCysIlePheHisProGluAlaProAlaCysLeuHis                              505560                                                                        ProAlaAspAlaGlnCysThrPheAlaSerProTyrArgSerGluThr                              65707580                                                                      ValTyrSerArgLeuTyrGluGlnCysArgProAspProAlaGlyArg                              859095                                                                        TrpProHisGluCysGluGlyAlaAlaTyrAlaAlaProValAlaHis                              100105110                                                                     LeuArgProAlaAsnAsnSerValAspLeuValPheAspAspAlaPro                              115120125                                                                     AlaAlaAlaSerGlyLeuTyrValPheValLeuGlnTyrAsnGlyHis                              130135140                                                                     ValGluAlaTrpAspTyrSerLeuValValThr                                             145150155                                                                     (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 155 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GluAlaIleLeuPheSerProGlyGluThrPheSerThrAsnValSer                              51015                                                                         IleHisAlaIleAlaHisAspXaaXaaAspGlnThrTyrSerMetAsp                              202530                                                                        ValValTrpLeuArgPheAspValProThrSerCysAlaGluMetArg                              354045                                                                        IleTyrGluSerCysLeuTyrHisProGlnLeuProGluCysLeuSer                              505560                                                                        ProAlaAspAlaProCysXaaXaaAlaAlaSerThrTrpThrSerArg                              65707580                                                                      LeuAlaValArgSerTyrAlaGlyCysSerArgThrAsnProXaaXaa                              859095                                                                        XaaProProArgCysSerAlaGluAlaHisMetGluProValProGly                              100105110                                                                     LeuAlaTrpGlnAlaAlaSerValAsnLeuGluPheArgAspAlaSer                              115120125                                                                     ProGlnHisSerGlyLeuTyrLeuCysValValTyrValAsnAspHis                              130135140                                                                     IleHisAlaTrpGlyHisIleThrIleSerThr                                             145150155                                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 208 nucleotides                                                   (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GAGCGGCCCGACCGCCGCCGGGTTGTTAAATGGGTCTCGCGCGGCTCGTG50                          GTTCCACACCGCCGGAGAACCAGCGCTGCGAGGGGGGGCTTGGTGGCTGG100                         CGACTCTTTAAGGCGTGCCGCCACGAGCAAGAAGACGGCCTGTATGCTAT150                         GCTCCCGCCGGACTATTTTCCGGTGGTGCCCTCGTCCAAGCCCCTGCTGG200                         TGAAAGTT208                                                                   (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 nucleotides                                                    (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGCACCGGTCCCGGATGCGAGGGGGGGCTTGGCCGGAGAACCAGCGCTGC50                          GAGGGGGGGCTTGG64                                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 nucleotides                                                    (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCGGAGAACCAGCGCTGCGAGGGGGGGCTTGGCCGGAGAACCAGCGCGAG50                          CTTCGCTGCGTGTG64                                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 756 nucleotides                                                   (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GGCCACGTGGAAGCTTGGGACTACAGCCTAGTCGTTACTTCGGACCGTTT50                          GGTGCGCGCGGTCACCGACCACACGCGCCCCGAGGCCGCAGCCGCCGACG100                         CTCCCGAGCCAGGCCCACCGCTCACCAGCGAGCCGGCGGGCGCGCCCACC150                         GGGCCCGCGCCCTGGCTTGTGGTGCTGGTGGGCGCGCTTGGACTCGCGGG200                         ACTGGTGGGCATCGCAGCCCTCGCCGTTCGGGTGTGCGCGCGCCGCGCAA250                         GCCAGAAGCGCACCTACGACATCCTCAACCCCTTCGGGCCCGTATACACC300                         AGCTTGCCGACCAACGAGCCGCTCGACGTGGTGGTGCCAGTTAGCGACGA350                         CGAATTTTCCCTCGACGAAGACTCTTTTGCGGATGACGACAGCGACGATG400                         ACGGGCCCGCTAGCAACCCCCCTGCGGATGCCTACGACCTCGCCGGCGCC450                         CCAGAGCCAACTAGCGGGTTTGCGCGAGCCCCCGCCAACGGCACGCGCTC500                         GAGTCGCTCTGGGTTCAAAGTTTGGTTTAGGGACCCGCTTGAAGACGATG550                         CCGCGCCAGCGCGGACCCCGGCCGCACCAGATTACACCGTGGTAGCAGCG600                         CGACTCAAGTCCATCCTCCGCTAGGCGCCCCCCCCCCCCCGCGCGCTGTG650                         CCGTCTGACGGAAAGCACCCGCGTGTAGGGCTGCATATAAATGGAGCGCT700                         CACACAAAGCCTCGTGCGGCTGCTTCGAAGGCATGGAGAGTCCACGCAGC750                         GTCGTC756                                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 nucleotides                                                    (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACGTGGTGGTGCCAGTTAGC20                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 nucleotides                                                    (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ACCAAACTTTGAACCCAGAGCG22                                                      __________________________________________________________________________

We claim:
 1. A mutant of bovine herpesvirus type 1 (BHV-1) having adeletion of the glycoprotein gE-gene, wherein said deletion allows themutant to be distinguished serologically from wild-type BHV-1.
 2. ABHV-1 mutant according to claim 1 wherein said deletion of the gE-genehas been caused by serial passage of the virus through cells wherebysaid BHV-1 mutant is obtained.
 3. A BHV-1 mutant according to claim 2which is Difivac-1 (Institut Pasteur, France, deposit No. I-1213).
 4. ABHV-1 mutant according to claim 1 which, in addition to said deletion ofthe gE-gene, has a deletion of the thymidine kinase gene.
 5. A BHV-1mutant according to claim 1 which, in addition to said deletion of thegE-gene, has a deletion of the glycoprotein gI-gene.
 6. A BHV-1 mutantaccording to claim 1 which, in addition to said deletion of the gE-gene,has a deletion of the thymidine kinase gene and a deletion of thegI-gene.
 7. A BHV-1 mutant having a deletion of the glycoproteingE-gene, wherein said deletion allows said mutant to be distinguishedserologically from wild-type BHV-1 by a process of discriminatingbetween BHV-1 viruses having an intact gE-gene and BHV-1 viruses havinga deletion of the gE-gene, said process comprising the step of examiningwhether nucleic acid of the virus reacts with gE specific probes orprimers derived from the nucleotide sequence coding for gE.
 8. A BHV-1mutant having a deletion of the glycoprotein gE-gene, wherein saiddeletion allows said mutant to be distinguished serologically fromwild-type BHV-1 by a process of discriminating between BHV-1 virusesexpressing gE and BHV-1 viruses having a deletion of the gE-gene, saidprocess comprising the step of examining whether the virus reacts withgE-specific antibodies raised against gE or against peptides derivedfrom the amino acid sequence of gE.
 9. A vaccine composition for avaccination of animals, in particular mammals, more in particularbovines, to protect them against BHV-1, wherein the vaccine compositionis a live or an inactivated vaccine comprising a BHV-1 mutant accordingto claim 1, and a suitable carrier or adjuvant.